Analysis methods for multiplex tissue imaging including imaging mass cytometry data

Abstract

The invention relates to methods for multiplex tissue imaging by methods such as imaging mass cytometry (IMC) and methods for analysis of imaging mass cytometry data. In various embodiments, methods are provided of cell-of-origin analysis and mutational analysis, coupled with spatial parameters derived from tumor clusters in the tumor microenvironment; which reveals signature marker profiles and therapeutic targets for treating cancers including diffuse large B cell lymphoma.

1 . A method of performing complex spatial analysis of a tissue sample, comprising:
 identifying one or more phenotypic clusters of cells relative to an index cell in a tissue sample or an image of the tissue sample, wherein each phenotypic cluster is identified based on two or more neighboring cells of a same phenotype; and   measuring a marker intensity of each phenotypic cluster of cells.   
     
     
         2 . The method of  claim 1 , wherein the tissue sample is from a subject diagnosed with a cancer, suspected of having a cancer, or having a refractory or relapsed cancer; and the method identifies one or more phenotypic clusters of cancer cells and one or more types of immune cells, macrophages and endothelial cells in the tissue sample. 
     
     
         3 . The method of  claim 1 , wherein the tissue sample comprises a tumor tissue selected from the group consisting of diffuse large B cell lymphoma (DLBCL), Hodgkin's lymphoma, other non-Hodgkin lymphoma, breast cancer, ovarian cancer, prostate cancer, melanoma, and a combination thereof. 
     
     
         4 . The method of  claim 1 , wherein the index cell is a tumor cell, and the marker comprises one or more immune cell markers or macrophage markers; or wherein the index cell is an immune cell, and the marker comprises one or more tumor cell markers. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , further comprising calculating a centroid location of each phenotypic cluster of cells by averaging the X,Y coordinates of the two or more neighboring cells of the same phenotype, and calculating a centroid distance between the centroid location of each phenotypic cluster to the index cell; optionally further comprising standardizing the distances between the index cell and each centroid location by dividing each centroid distance by the total number of cells of the corresponding phenotype. 
     
     
         8 . The method of  claim 7 , wherein the index cell is an immune cell and each cluster is a tumor phenotype, and the method further comprising:
 identifying a first, tumor-core-immune-desert zone which comprises a phenotypic cluster whose centroid distance to the index cell is no less than a first threshold or the farthest in all clusters, or within which immune activity is lowest in all clusters as characterized by substantially absent of proliferative CD8+ T cells, macrophages or T REG  cells;   identifying a second, tumor-dispersed-immune-activation zone which comprises a phenotypic cluster whose centroid distance to the index cell is no greater than a second threshold or the shortest in all clusters, or within which immune activity is activated as characterized by substantial presence of proliferative CD8+ T cells, macrophages and T REG  cells; and/or   identifying a third, tumor-immune-interface zone which comprises a phenotypic cluster whose centroid distance to the index cell is between the first threshold and the second threshold or between the first zone and the second zone, or within which immune activity is suppressed as characterized by substantial presence of exhausted CD8+ T cells and suppressive T REG  cells.   
     
     
         9 . The method of  claim 8 , wherein the first threshold is 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm or 47 μm, and the second threshold is 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 8 , further comprising performing a mutation screen of a plurality of genes on the tissue sample, wherein gene mutations of cells within at least one of the zones are identified; and/or further comprising performing cell-of-origin analysis on the tissue sample by profiling gene expressions in the cells, wherein B cells within at least one of the zones are identified as one of germinal center B-cell (GCB), activated B-cell (ABC), double-expressor, and double-hit or triple-hit lymphoma. 
     
     
         12 . The method of  claim 11 , wherein the plurality of genes comprises BCL2, BCL6, EZH2, CD79, MYD88 L265P , TP53, SGK1, 9p24 Gain, NOTCH1, NOTCH2, or a combination of any two, three, four, five, six, seven, eight, nine or ten genes thereof. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the two or more neighboring cells comprise 5-10, 11-15, 16-20, 21-25, or 26-30 neighboring cells, each being adjacent to at least one other cell in the cluster, and the method identifies 2-4, 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, or 46-50 different phenotypic clusters of cells; and wherein the two or more neighboring cells of the same phenotype are the closest in distance of that phenotype to the index cell. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1 , further comprising first performing an imaging mass cytometry (IMC) to generate an image of the tissue sample, or wherein the tissue sample is first subjected to mass spectrometry imaging to generate an image of the tissue sample, wherein measuring a marker intensity includes measuring a plurality of markers comprising any one, partial combination, or all 32 antigens or markers according to Table 2, and optionally the cells of the tissue sample comprise a label selected from the group consisting of an antibody label, an isotope label, a fluorescent label, a fluorochrome label, a fluorophore label, and combinations thereof. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A method for profiling a tumor microenvironment, comprising:
 performing imaging mass cytometry (IMC) on a tumor sample from a subject in need thereof, thereby obtaining imaging mass cytometry data of the tumor sample; and   performing a method of complex spatial analysis according to  claim 8  on the tumor sample.   
     
     
         21 . The method of  claim 20 , further comprising providing a prognosis for survival of the subject, and/or selecting or administering a treatment for the subject, based on findings in the complex spatial analysis; wherein the subject has a cancer selected from the group consisting of diffuse large B cell lymphoma (DLBCL), Hodgkin's lymphoma, breast cancer, ovarian cancer, prostate cancer, melanoma, and combinations thereof. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A method of identifying and treating a subject with diffuse large B cell lymphoma (DLBCL), comprising:
 detecting an increased amount of TIM-3+ T cells in a lymphoma sample of the subject relative to a control, said control comprising a subject with Hodgkin's lymphoma or with no lymphoma,   administering to the subject an effective amount of an anti-TIM-3 antibody.   
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 24 , further comprising administering to the subject an effective amount of engineered immune cells which expresses chemokine receptor CXCR3; and wherein the method does not include administering a PD-1 antagonist to the subject. 
     
     
         27 . (canceled) 
     
     
         28 . A method of predicting response to a therapy in a subject having a cancer, comprising performing complex spatial analysis on a tissue sample of the subject according to  claim 8 , wherein the marker intensity of at least one of the phenotypic cluster is indicative of response to the therapy; and
 wherein if the therapy comprises a chemotherapy, then the subject's response to the chemotherapy is indicated as ineffective if:
 the cancer cells express a combination of PD-L1, TIM-3 and CCR4, 
 suppressive TREG or PD-L1+M2 macrophages are present in the cancer cells' microenvironment, 
 a largest tumor cell cluster expresses PD-L1, or 
 a largest tumor cell cluster expresses Ki67 and CCR; 
   or   wherein if the therapy comprises a drug that targets PD-1, then the subject's response to the drug that targets PD-1 is indicated as ineffective if there is an increased presence of TIM-3−PD-L1+ macrophages and/or TIM-3+ T cells within microenvironment of malignant B-cells.   
     
     
         29 . The method of  claim 28 , wherein the complex spatial analysis is performed from a tissue sample collected from the subject after initiation of the therapy. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . A method for calculating an intensity-weighted abundance score (M-score) of a marker in a tissue sample, comprising:
 measuring a normalized marker intensity across all cells in the tissue sample or an image of the tissue sample;   dividing the tissue sample or the image of the tissue sample into a plurality of blocks based on quantiles of a total dynamic range of the tissue sample or the image of the tissue sample;   assigning an average intensity of cells in each block of the plurality of blocks; and   counting a number of cells in each block of the plurality of blocks;   wherein the intensity-weighted abundance score of the marker is obtained by a linear combination of the number of cells in each block and the average intensity of the respective block.   
     
     
         33 . The method of  claim 32 , further comprising first using imaging mass cytometry (IMC) to generate an image of the tissue sample, or wherein the tissue sample is first subjected to mass spectrometry imaging to generate an image of the tissue sample; wherein optionally the cells are labeled with a label selected from the group consisting of an antibody label, an isotope label, a fluorescent label, a fluorochrome label, a fluorophore label, and combinations thereof. 
     
     
         34 . The method of  claim 32 , wherein the plurality of blocks is 2-5, 6-10, 10-15, or 16-20 blocks, and wherein the intensity-weighted abundance score is calculated for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, or 71-75 markers in the tissue sample. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled)